Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a 13C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg2+ ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn2+ or Cd2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2′-OCH3-U5 modified twister ribozyme.

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pK a value of 5.1 was determined for a 13 C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pK a of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg 2+ ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn 2+ or Cd 2+ accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2′-OCH 3 -U5 modified twister ribozyme. Keywordsmetal ion rescue; nucleoside modifications; oligoribonucleotides; perturbed pK a ; solid-phase synthesis Small self-cleaving ribozymes are widely distributed in nature [1] and are essential for rolling-circle-based replication of satellite RNAs. [2,3] Among them, the hepatitis delta virus (HDV) ribozyme [4][5][6][7][8] employs a divalent cation in the active site for catalysis, while the remaining small self-cleaving ribozymes including hammerhead, [2,9,10] hairpin, [3,[11][12][13] glmS, [14][15][16] and Varkud Satellite [17] employ principles of general acid-base and electrostatics for catalysis. Very recently, a new class of nucleolytic ribozymes (termed twister) has been discovered, [18] and soon thereafter, crystal structures were published that revealed a common double-pseudoknot overall architecture for the twister ribozyme but showed clear distinctions in residue and divalent cation alignments at the cleavage site. [19][20][21] While the O. sativa twister ribozyme was off-line orthogonally aligned with a fully base-paired stem P1, [19,20] the env22 twister ribozyme was in-line oriented at the cleavage step A6-U5, with a Mg 2+ coordinated to the scissile phosphate. Furthermore, for the env22 twister ribozyme, stem P1 formed only the two central base pairs (Figure 1) while the neighboring nucleotides U1 and U4 were instead engaged in stacked base triplet interactions (U4-A49-A34 and U1-A50-U33; Figure 1B). [21] Košutić et al.Page 2 Angew Chem Int Ed Engl. Author manuscript; available in PMC 2016 January 16. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThese contrasting observations were the starting point for the present investigation. A thorough comparison of the two structures (PDB: 4OJI for O. sativa and PDB: 4RGF for env22) revealed that the conserved adenosine (A6; env22 numbering is used throughout) at the cleavage site adopts nearly identical conformations involving ex...

Methylphosphonate(mP)-modified RNA serves as valuable probe to evaluate biomolecular interactions between the nucleic acid backbone and binding partners, such as proteins or small molecules. Here, we describe an efficient workflow for the synthesis of RNA with a single mP modification in diastereomerically pure form. While the automated assembly of mP-modified RNA is straightforward, its deprotection under basic conditions is challenging; a carefully optimized step-by-step procedure is provided. In addition, we demonstrate purification and separation strategies for the RP and SP-configurated RNA diastereomers using a combination of anion-exchange and reversed-phase HPLC, and comment on troubleshooting if their separation appears difficult. Furthermore, we demonstrate the stereochemical assignment of short RP and SP mP-modified RNA diastereomers based on 2D ROESY NMR spectroscopy and we report on the impact of the mP modification on thermal and thermodynamic stabilities of RNA-DNA hybrid and RNA-RNA duplexes.

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